Effect of Pollen Protein Diffusates on Germination of Eluted Pollen Samples of Petunia hybrida in vitro

Germination in vitro of pollen grains of Petunia hybrida L.is sharply reduced by brief elution with cold distilled water.If eluted substances are added back to eluted pollen germinatingin vitro, the germination capacity is significantly restored.A heat-labile protein fraction (50000–100000 daltons)is responsible for restoring the germination ability. Petunia hybrida L, pollen, protein, diffusates, germination     

Detection of Pollen Germination Inhibitors in Brassica oleracea Tissue Extracts

Petunia hybrida and Lilium lankongense pollens were germinatedon thin layer chromatography (TLC) plates following chromatographyof extracts from the self-, cross- and unpollinated stigmas,styles and ovaries and the seeds, leaves and pollen of threeinbred Brassica oleracea families. Zones of pollen germinationinhibition on the TLC plates showed that inhibitory compoundswere present in the tissue extracts. The R_f values and numberof these compounds varied with the tissue used, stigma tissuecontaining the largest amounts and the greatest number of inhibitors.In contrast, differences between the inbred lines tested wereslight and quantitative. Pollen from both P. hybrida and L.lankongense gave the same results; that from B. oleracea couldnot be used because of its poor germination. Brassica oleracea, Brussels sprout, kale, Lilium lankongense, Petunia hybrida, pollen germination, thin layer chromatography, germination inhibitors, phytoalexin, bioassay     

The Effect of Brassica oleracea Stigma Extracts on the Germination of B. oleracea Pollen in a Thin Layer Chromatographic Bioassay

Hodgkin, T. and Lyon, G. D 1986. The effect of Brassica oleraceastigma extracts on the germination of B. oleracea pollen ina thin layer chromatographic bioassay.—J. exp. Bot. 37:406–411. A procedure for germinating Brassica oleracea pollen on thinlayer chromatography plates pretreated with 20 mol m^–3tris(hydroxymethyl) methyl-aminopropanesulphonic acid (TAPS)buffer, pH 8·0 has been devised and used to detect pollengermination inhibitors in B. oleracea stigma extracts. Inhibitory zones in extracts of stigmas, unpollinated, or collected0·5, 4, 8 and 24 h after self- or cross-pollination,differed little in R_F values and sizes. Extracts of stigmascollected 1 h and 2 h after self-pollination gave a small additionalinhibitory zone which was not detected in 1 h and 2 h cross-pollinatedstigma extracts. The results showed some differences from thoseobtained using Petunia hybrida pollen germinated on T.L.C. platesthat were not pretreated with buffer. The nature of the differencesbetween the two bioassays is discussed and some possible reasonsfor them indicated. Key words: Pollen, germination inhibitors, self-incompatibility, Brassica oleracea     

The Changes of Protein Content and Synthetic Activity in Squash Pollen During Germination

The total protein content of squash (Cucurbita moschata Duch.) pollen decreased gradually during in vitro germination. It was caused by the release of wall proteins and part of the cytoplasmic proteins. The release of the pollen wall proteins was not dependent on germination, it was a passive diffusion process. However, the cytoplasmic proteins did not release until the pollen germinated, a fraction of them was synthesized de novo during germination. The RNA and protein synthetic activities initiated soon after in vitro pollen germination. The RNA synthesis decreased during germination. As about half the activity was inhibited by α-amanitin, mRNA might be the major RNA synthesized de novo. The total protein synthesis increased during germination, almost all of this synthesis was inhibited by cycloheximide, and partially by α-amanitin, but it was not affected significantly by actinomycin D. These results indicated that both stored and de novo synthesized mRNA might play a role in the protein synthesis. The content of stored mRNA of squash pollen was about 11-3 pg/grain as measured by UV absorption after its purification from total RNA (2440 pg/grain) by oligo (dT)-cellulose affinity chromatagraphy. Both cycloheximide and α-amanitin inhibited pollen tube growth in vitro. Actinomycin D and tunicamycin inhibited pollen germination in the first hour, however, no reduction ,of the tube length was observed later. Cyclohex,nide inhibited the pollen germination and tube elongation in vivo, that fitted well with the in vitro results. According to these results, it was suggested that the de novo syntheses of mRNA and protein were neccessary for the maintenance of pollen tube growth.     

Evidence for DNA repair after ultraviolet irradiation of Petunia hybrida pollen

Summary Ultraviolet irradiation of Petunia hybrida pollen led to an unscheduled labelling of pollen DNA by ³H-thymidine during the early stages of germination. Hydroxyurea increased this DNA labelling, while added boron, required absolutely for pollen germination, tube elongation and tube generative cell mitosis, was not needed for this repair — like DNA synthesis.     

The influence of relative humidity on the swelling of pollen grains in vitro

The volume of hydrated pollen grains of Petunia hybrida L. during swelling in germination medium increased three times. The volume of desiccated pollen grains increased only two times after transfer to the same medium. This difference in swelling ability is attributed to different rigidities of the pollen grain wall,ccaused by the different hydration states. The relationship between pollen grain swelling and germination metabolism with regard to relative humidity is discussed.Abbreviation RH relative humidity     

RNA and Protein Synthesis in Germinating Pine Pollen

The results of autoradiographic experiments demonstrate that,as with the pollen of most other species, both the generativeand vegetative nuclei of Loblolly Pine (Pinus taeda) activelyengage in RNA synthesis from the very early stages of pollengermination. Unlike most other species, however, this newlysynthesized RNA includes rRNA. Evidence is provided for theimportance of this newly synthesized RNA in the process of continuedpollen tube growth. One and two-dimensional gel electrophoretic analysis revealsa number of both qualitative and quantitative differences amongthe proteins synthesized during the early stages of germinationand the later stages of pollen tube growth. One of the mostnotable of these is a 36 kD protein, the synthesis of whichpredominates during the later stages of pollen germination.A similar pattern of 36 kD protein synthesis is observed whenmRNA extracted from pollen at each of these stages is translatedin vitro. Key words: Pinus, pollen tube growth     

Stigma-surface Esterase Activity and Stigma Receptivity in Some Taxa Characterized by Wet Stigmas

Stigma-surface esterase activity and stigma receptivity througha sequence of developmental stages of the pistil have been studiedin four taxa characterized by having wet stigmas — Petuniahybrida, Nicotiana tabacum, Crinum defixum and Amaryllis vittata.The style is solid in the first two and hollow in the lattertwo taxa. In all the taxa, stigma—surface esterase couldbe detected in a thin surface layer (pellicle) from a very earlystage of pistil development, irrespective of the presence orabsence of the exudate. However, the taxa showed variation instigma receptivity. In Petunia and Nicotiana, stigmas from pistilsof all the stages supported pollen germination and tube growth.In Amaryllis and Crinum, stigmas of only the mature pistils,when the exudate is present on the stigma, supported normalpollen germination and tube growth. It is inferred that in taxacharacterized by a wet stigma and solid style, the factors requiredfor pollen germination are present from an early stage of pistildevelopment and the exudate per se is not involved in pollengermination. In taxa characterized by a wet stigma and hollowstyle, however, the pellicle does not carry the factors requiredfor pollen germination and tube growth; they appear to be presentin the exudate. Petunia hybrida Vilm, Nicotiana tabacum L., Crinum defixum, Ker-Gawl, Amaryllis vittata Ait., tobacco, pollination, pollen germination, stigmatic exudate, stigma receptivity, stigma-surface esterase, esterase activity     

Pollen DNA repair after treatment with the mutagens 4-nitroquinoline-1-oxide, ultraviolet and near-ultraviolet irradiation, and boron dependence of rapair

Summary Irradiation of dry, mature pollen from Petunia hybrida with near-ultraviolet light from an erythemal-sunlamp gave rise to a repair-like, unscheduled DNA synthesis during the early stages of in vitro germination. Like that brought about by farultraviolet light from a germicidal lamp, this DNA synthesis is enhanced by hydroxyurea added to the germination medium, and reduced by photoreactivating light given after ultraviolet irradiation and before germination begins. It is concluded that pollen, often receiving considerable exposure to sunlight, has, in addition to the protection afforded by the ultraviolet filtering effect of yellow pigments, also the capacity to repair ultraviolet produced changes in DNA, by both photoreactivation and dark repair processes.Because mature Petunia pollen is arrested at the G₂ stage of the cell cycle, germinating pollen provides us with a highly synchronous plant tissue with a very low background of DNA replicative synthesis suitable for sensitive measurement of DNA repair synthesis. Thus we have shown that 4-nitroquinoline-1-oxide, at concentrations greater than 0.001 mM, gives rise to an unscheduled DNA synthesis which is enhanced by hydroxyurea. Like that induced by ultraviolet radiation, the chemical mutagen brings about DNA repair only during the early stages of pollen germination, and further it has been possible to show that repair ceases at about the time that generative cell division and pollen tube elongation begins.Boron addition enhances both ultraviolet and 4-nitroquinoline-1-oxide induced repair synthesis. By delaying the chemical mutagen initiation of repair until after germination has begun, we have been able to show that boron is most beneficial during the first hour of germination. It is postulated that this is achieved through an as yet unknown effect of boron on the supply of precursors before pollen cell metabolism is fully committed to pollen tube synthesis later in the germination period.     

Overcoming Self-incompatibility through the Use of Lectins and Sugars in Petunia and Eruca

Treatment of stigma with a lectin (Con A/PHA) before pollinationwas effective in overcoming self-incompatibility in Petuniahybrida, a gametophytic self-incompatible system, and Erucasativa, a sporophytic self-incompatible system. Treatment ofpollen with glucose/N-acetyl-D-galactosamine (tested only withPetunia) was also effective. These results suggest the involvementof pollen lectins and specific sugar components of the pistilin self-incompatibility recognition. Petunia hybrida, Eruca sativa, self-incompatibility, pollen recognition, lectins     

Flavonols stimulate development, germination, and tube growth of tobacco pollen

The effect of anther-derived substances on pollen function was studied using pollen produced by in vitro culture of immature pollen of tobacco (Nicotiana tabacum L.) and petunia (Petunia hybrida). Addition of conditioned medium consisting of diffusates from in situ matured pollen strongly increased pollen germination frequency and pollen tube growth, as well as seed set after in situ pollination. Thin-layer chromatography and depletion of phenolic substances by Dowex treatment indicated that flavonols are present in the diffusate and may be the active compounds. When added to the germination medium, flavonols (quercetin, kaempferol, myricetin) but not other flavonoids strongly promoted pollen germination frequency and pollen tube growth in vitro. The best results were obtained at very low concentrations of the flavonols (0.15-1.5 μm), indicating a signaling function. The same compounds were also effective when added during pollen development in vitro.     

Purine Nucleoside Transport in Petunia Pollen Is an Active, Carrier-Mediated System Not Sensitive to Nitrobenzylthioinosine and Not Renewed during Pollen Tube Growth

Adenosine and guanosine are transported into Petunia hybrida pollen by a saturable, carrier-mediated mechanism. The energy poisons carbonylcyanide-m-chlorophenylhydrazone, 2,4-dinitrophenol, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, and N,N′-dicyclohexylcarbodiimide all inhibit uptake, suggesting an energy coupled (active) transport process. Transport takes place against a concentration gradient, strongly favoring an active transport mechanism. The purine nucleoside transport in Petunia pollen differs from that already reported for pyrimidine nucleosides in that it exhibits a significantly higher K_m for nucleoside and is not so severely inhibited by the polyamine, spermine. Like that for the pyrimidine nucleosides uridine and cytosine, however, the system exhibits a broad pH optimum, is inhibited by sulfydryl-binding reagents, while the potent inhibitors of nucleoside transport in animal cells, nitrobenzylthioinosine and dipyridamole, have no effect. Transport of both purine and pyrimidine nucleosides in germinating pollen decreases steadily with time, a finding consistent with reports that RNA synthesis and DNA repair are early events of pollen germination and tube elongation. However, since these precursors are often used to demonstrate nucleic acid synthesis, it cannot be ruled out that the lack of precursor transport itself leads to scoring nucleic acid synthesis as negative. The results indicate that the newly synthesized pollen tube membranes contain little or no nucleoside transporters.     

Pollen Tube Development and Characteristics of the Protein Emission in Conifers

When ripe, viable pollen of Pinus contorta, Pinus nigra, Piceasitchensis, Abies alba and Cedrus deodara is germinated on asuitable artificial substrate, the process of pollen tube growthbegins 12–36 h later, according to the species. The tubeemerges at the leptoma, a structurally specialized site in thepollen wall, and the early tube wall is continuous with thetwo microfibrillar intine strata within the grain. Later inpollen tube development, when cytoplasmic zonation has beenestablished, only the inner of these two wall layers is differentiated. Cytochemical methods show that, during hydration and throughoutthe period of tube growth in culture, proteins are releasedfrom the pollen grains; before germination most conspicuouslyfrom the leptoma, subsequently from the tube itself. The emissioncontains a number of hydrolytic enzymes and a non-catalyticmoiety. Gel-electrofocusing reveals that the hydrolases releasedfrom germinating Pinus contorta pollen include several acidphosphatase and esterase isozymes, and that there are differencesbetween the composition of the emission and the spectrum ofsoluble proteins extracted from the pollen grains before germination.Analysis by immunodiffusion demonstrates that two componentswith antigenic characteristics present in the quiescent pollengrains are represented in the emission. The possible utilityof the released components in the biology of conifer reproductionis discussed. Pinus contorta, Pinus nigra, Picea sitchensis, Abies alba, Cedrus deodara, lodgepole pine, Austrian pine, Sitka spruce, European silver fir, deodar, pollen tube, pollen germination, protein release, isoelectric focusing     

Male Sterility and Anther Wall Structure in Copper-deficient Plants

Anther development and pollen sterility were followed in plantsof wheat, oat, barley, sweetcorn, sunflower, petunia and subterraneumclover grown at a range of copper supplies. Copper-deficientplants had increased pollen sterility. Lignified wall thickenings were reduced or absent in the endotheciaof anthers from Cu-deficient plants. Reduced seed set may resultboth from reduced pollen fertility or failure of the stomiato rupture due to decreased lignification of anther walls. Triticum aestivum L., wheat, Hordeum vulgare L., barley, Avena sativa L., oat, Zea mays L., corn, sweetcorn, maize, Helianthus annuus L., sunflower, Petunia hybrida L., Trifolium subterraneum L., subterranean clover, male sterility, anther development, copper deficiency     

EFFECT OF SALINITY ON POLLEN I. POLLEN VIABILITY AS ALTERED BY INCREASING OSMOTIC PRESSURE WITH NaCl,MgCl2, and CaCl2

Petunia (Petunia hybrida Vilm. cv. ‘Snowstorm') plants were grown in saline solution (NaCl, MgCl₂, and/or CaCl₂) of 0, 1, 2, and 3 bars osmotic pressures. Pollen viability was tested by tetrazolium chloride staining and by germination (by the hanging drop method, using 15 % sucrose and 0.01 % boric acid as the nutrient medium, at 27 ± 1 C). Pollen viability decreased with increased salinity. Pollen from plants grown in single salt solutions of NaCl, MgCl₂, and CaCl₂ (each at 0, 1, 2, or 3 bars osmotic pressure) was germinated in base culture medium. Pollen viability decreased more with NaCl than with MgCl₂ or CaCl₂. In vitro studies of the effects of three salts, viz., NaCl, MgCl₂, and CaCl₂, on pollen germination and tube growth showed that NaCl inhibited germination and pollen tube growth more than did MgCl₂ or CaCl₂. MgCl₂ was least injurious, and even promoted tube growth at 0.5 and 0.75 bars osmotic pressure. Adding low concentrations of MgCl₂ reduced the toxic effect of NaCl and increased the percentage of germination. CaCl₂ reduced the effect of NaCl less than did MgCl₂. We conclude that specific ion effects were more important than osmotic pressure.     

Pollen Diffusates of Crotalaria retusa and their Role in pH Regulation

Details of the release of proteins and amino acids from culturedpollen grains and the role of the leached metabolites in pollengermination, pollen tube growth and regulation of pH of theculture medium in Crotalaria retusa have been investigated.In unbuffered media, satisfactory pollen germination and tubegrowth occurred over a wide range of pH values 4.0–9.0.This was related to the ability of pollen diffusates to shiftthe pH to 6.25 in all these media. Similar pollen germinationand pH shift was observed when the pollen was eluted twice beforeculturing. When the pH shift was reduced by using buffered media,optimal germination and tube growth occurred only at pH 6.0.Pollen diffusates had a strong buffering capacity. Proteinsand amino acids released from pollen do not seem to have a directrole in pH regulation. The components involved in pH regulationmay originate from the pollen wall as well as from the cytoplasm. Crotalaria retusa L, pH regulation, pollen diffusates, pollen germination     

Regulation der Nucleinsäuren-Synthese durch Polyamine in keimendem Pollen vonPetunia

Summary Putrescine, spermine, spermidine, and agmatine in concentrations between 5–15 g/ml inhibit pollen germination. Whereas spermine reduces pollen tube length, putrecine and agmatine do not affect pollen tube growth. Spermidine effects a small increase (about 5%) of pollen tube elongation. Spermine and spermidine can be found in pollen. Addition of spermine (7 or 10 g/ml) depresses protein synthesis, whilst spermidine does not affect protein synthesis. On the basis of uridine-5-T incorporation it could be shown that both spermine and spermidine increase RNA synthesis. On tho basis of thymidine-T incorporation in the first hpurs of germination it seems that DNA synthesis is also stimulated by spermine and spermidine present in the medium. A net increase of nucleic acids was found when spermidine was added to the germination substrate.These results are interpreted as suggesting that, in the pollen tubes investigated, polyamine concentration may be a factor in the regulation of nucleic acid synthesis, resulting in a prolonged synthesis of specific proteins and in this way influencing growth and the developmental pattern of pollen tubes.     

The role of abscisic acid in germination, storage protein synthesis and desiccation tolerance in alfalfa (Medicago sativa L.) seeds, as shown by inhibition of its synthesis by fluridone during development

Abscisic acid and osmoticum maintain maturation and proteinsynthesis of developing alfalfa embryos, individually and incombination. The in situ environment of developing alfalfa zygoticembryos is rich in ABA and low in osmotic potential. When ABAsynthesis was inhibited by treating the pods with fluridoneat an early stage of development, the seeds which subsequentlydeveloped contained low amounts of ABA, but had a similar osmoticpotential as untreated control seeds. The reduced ABA in seedsfrom fluridone-treated pods did not change the morphology exceptthe colour of seeds, nor did it induce viviparous germinationor affect storage protein synthesis. However, two nonstorageproteins which were synthesized in control seeds during earlyto mid-development were absent from fluridone-treated seeds.Control seeds containing these two proteins were desiccation-tolerant,whereas the fluridone-treated seeds which lacked them were desiccation-intolerant,at least until the deposition of storage proteins was nearlycomplete. Culture of isolated embryos on nutrient medium inducedgermination and curtailed storage protein synthesis in the embryos.Addition of either ABA or osmoticum to the nutrient medium preventedgermination and maintained storage protein synthesis. When fluridonewas added along with osmoticum, germination occurred, but storageprotein synthesis was maintained. Key words: Embryogenesis, Medicago sativa L., alfalfa, ABA, osmotic potential, fluridone, desiccation, storage protein synthesis     

Gene Activity during Germination of Spores of the Fern, Onoclea sensibilis: RNA and Protein Synthesis and the Role of Stored mRNA

Pattern of ³H-uridine incorporation into RNA of spores of Onocleasensibilis imbibed in complete darkness (non-germinating conditions)and induced to germinate in red light was followed by oligo-dTcellulose chromatography, gel electrophoresis coupled with fluorographyand autoradiography. In dark-imbibed spores, RNA synthesis wasinitiated about 24 h after sowing, with most of the label accumulatingin the high mol. wt. poly(A)^–RNA fraction. There was noincorporation of the label into poly(A) ⁺ RNA until 48 h aftersowing. In contrast, photo-induced spores began to synthesizeall fractions of RNA within 12 h after sowing and by 24 h, incorporationof ³H-uridine into RNA of irradiated spores was nearly 70-foldhigher than that into dark-imbibed spores. Protein synthesis,as monitored by ³H-arginine incorporation into the acid-insolublefraction and by autoradiography, was initiated in spores within1–2 h after sowing under both conditions. Autoradiographicexperiments also showed that the onset of protein synthesisin the cytoplasm of the germinating spore is independent ofthe transport of newly synthesized nuclear RNA. One-dimensionalsodium dodecyl sulphate-polyacrylamide gel electrophoresis of³⁵S-methionine-labelled proteins revealed a good correspondencebetween proteins synthesized in a cell-free translation systemdirected by poly(A) ⁺RNA of dormant spores and those synthesizedin vivo by dark-imbibed and photo-induced spores. These resultsindicate that stored mRNAs of O. sensibilis spores are functionallycompetent and provide templates for the synthesis of proteinsduring dark-imbibition and germination. Key words: Onoclea sensibilis, fern spore germination, gene expression, protein synthesis, sensitive fern, stored mRNA